Somatostatin receptor 2 (SSTR2) expression is associated with better clinical outcome and prognosis in rectal neuroendocrine tumors

Somatostatin analogues have recently been used as therapeutic targets for metastatic or surgically unresectable gastroenteropancreatic (GEP) neuroendocrine tumors (NETs), and associated somatostatin receptor (SSTR) expression has been well demonstrated in most GEP NETs, with the exception of rectal NETs. SSTR2 immunohistochemical expressions were evaluated in 350 surgically or endoscopically resected rectal NETs and compared to clinicopathologic factors. SSTR2 expression was observed in 234 (66.9%) rectal NET cases and associated tumors with smaller size (p = 0.001), low pT classification (p = 0.030), low AJCC tumor stage (p = 0.012), and absence of chromogranin expression (p = 0.009). Patients with rectal NET and SSTR2 expression had significantly better overall survival than those without SSTR2 expression both by univariable (p = 0.006) and multivariable (p = 0.014) analyses. In summary, approximately two-thirds of rectal NETs expressed SSTR2. SSTR2 expression was significantly associated with favorable behavior and good overall survival in patients with rectal NETs. Furthermore, SSTR2 expression can be used as prognostic factors. When metastatic disease occurs, SSTR2 expression can be used a possible target for somatostatin analogues.


Pathologic study
Pathologic data, including tumor size, location, depth of invasion, mitotic count, Ki-67 labeling index, lymphovascular and perineural invasion, lymph node and distant metastasis, resection marginal status and immunohistochemical results of synaptophysin and chromogranin expression, was reviewed.Cases were classified as NET grade 1 and grade 2 according to 2019 WHO classification on the basis of mitotic count and Ki-67 labeling index 5 .The TNM stage was classified based on the 8th American Joint Committee on Cancer (AJCC) cancer staging manual 31 .

Tissue microarray and immunohistochemistry
All hematoxylin and eosin slides of each case were reviewed and representative slides and paraffin blocks were retrieved.Tissue microarrays (TMAs) were constructed form representative paraffin blocks with tissue microarrayer (Uni TMA Co Ltd, Seoul, Republic of Korea).One 2.0 mm core was punched from donor tumor blocks and replaced into recipient blocks.Immunohistochemical staining was performed as previously described 6 .Briefly, from each TMA block, 4 μm thick sections were cut and deparaffinized and hydrated in xylene and ethanol.Endogenous peroxidase was blocked and heat-induced antigen retrieval was done.Sections were incubated with primary rabbit monoclonal antibody for SSTR2 (1:6400, ab134152, Abcam, Cambridge, UK).An OptiView DAB Detection Kit (Ventana Medical Systems) was used for the brown chromogen of SSTR2.Slides were counterstained with hematoxylin and dehydrated with ethanol.The result of SSTR2 immunohistochemical staining was graded into 4 groups on the basis of the extent of membranous staining (0, ≤ 5%; 1+, 6-25%; 2+, 26-50%; 3+, 51-75%; 4+, ≥ 76%).The cases were reclassified to 0 as negative group and 1+, 2+, and 3+ as positive groups as previously described 6,29 .

Statistical analyses
Chi-squared test and Fisher's exact test were performed to analysis the association between SSTR2 expression and clinicopathologic factors.The overall and recurrence free survival was evaluated with the Kaplan-Meier analysis with the log-rank test.The prognostic significance of SSTR2 was evaluated using the Cox proportional hazards regression model.P value < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).

SSTR2 expression in rectal NETs
Representative images of SSTR2 expression in normal rectal mucosa and rectal NETs are illustrated in Figs. 1 and  2. Peritumoral non-neoplastic colonic mucosa was evaluated in 83 cases and none of them showed SSTR2 expression.In contrast, SSTR2 expression was observed in 234 (66.9%) rectal NETs.SSTR2 expression was significantly associated with small tumor size (p = 0.001), low pT classification (p = 0.030), low AJCC stage (p = 0.012), and absence of chromogranin expression (p = 0.009; Table 1).www.nature.com/scientificreports/

Survival analyses according to SSTR2 expression in rectal NETs
The overall survival of rectal NET patients with SSTR2 expression was significantly better than those without SSTR2 expression [hazard ration (HR) 0.346; 95% confidential interval (CI) 0.157-0.759;p = 0.006].The 5-year survival rate of rectal NET patients with SSTR2 expression was significantly better than those without SSTR2 expression (98.5% vs. 92.6%,p = 0.006; Fig. 3a).Subgroup analysis based on tumor grade showed that grade 1 www.nature.com/scientificreports/rectal NET patients with SSTR2 expression had significant better overall survival than those without SSTR2 expression (5-year survival rate, 98.4% vs. 93.4%,p = 0.011; Fig. 3b).In contrast, the overall 5-year survival rate of grade 2 rectal NET patients with SSTR2 expression was better than those without SSTR2 expression though statistically not significant (100.0%vs. 85.7%, p = 0.196; Fig. 3c).2).Multivariable analyses were conducted with factors that were shown by the univariable analyses to be significant (Table 3).SSTR2 expression (p = 0.014), low tumor grade (p = 0.002), and absence of distant metastasis (p < 0.001) were an independent prognostic factors in rectal NET patients (Table 2).

Discussion
SSAs have been used to treat NETs, and radiolabeled SSAs have been increasingly used for imaging and therapy [31][32][33][34] .The expression of SSTR in NETs is the rationale for these clinical applications 35 .Among five subtypes of the SSTRs, SSTR2 is the most commonly expressed subtype in the NETs [36][37][38] .SSTR2 expression has previously been reported in GEP NETs, but it has not been specifically reported in rectal NETs.To the best of our knowledge, this is the first study on SSTR2 expression and its clinicopathologic correlation in large cohort of rectal NETs, including patients' survival.
SSTR2 expression was identified in approximately 70% of rectal NETs in the present study.Previous studies demonstrated that SSTR2 expression in a small cohort of rectal NETs (range 3-13 patients) and the majority of them were included as a component of left colon or colorectal NET cohorts 18,[38][39][40][41] .The proportion of SSTR2 expression in rectal NETs ranged from 14 to 100% in the previous studies 18,38,39,41 .The prevalence (70%) of SSTR2 expression in rectal NETs in the present study is similar with that of Oana et al., and they reported 53.8% (7 of www.nature.com/scientificreports/13 cases) of rectal NETs with SSTR2 expression 39 .Previously reported prevalence of SSTR2 expression in rectal NETs is variable 18,38,39,41 .Surprisingly, one study reported 3 rectal NETs with 100% SSTR positivity 41 .Hirofumi et al. reported SSTR2 expression in 10 out of 71 (14.1%) rectal NETs 40 .The low prevalence of SSTR2 expression in previous study may be due to different proportion of high grade (grade 2 and 3) rectal NETs.The present study include 324 (92.6%) cases of grade 1 and 26 (7.4%) cases of grade 2 rectal NETs, while previous study include 51 (71.8%) cases of grade 1 and 20 (28.2%) cases of grade 2 and 3 rectal NETs 40 .SSTR2 expression in rectal NETs was significantly correlated with favorable clinicopathologic factors, such as small size, absence of lymph node metastasis, low pT classification, low AJCC stage group, and negative chromogranin immunohistochemical expression.In addition, SSTR2 expression was significantly associated with favorable survival and an independent good prognostic factor in rectal NET patients.There have been a few studies of SSTR2 expression and compared their clinicopathologic correlation, including patients' survival in the pancreatic NETs, but no previous large cohort studies in rectal NETs.SSTR2 expression was significantly www.nature.com/scientificreports/correlated with improved survival rate and an independent good prognostic factor in previous studies with pancreatic NETs 26,27,42 , which was consistent with the present study.
To the best of our knowledge, this is the first large-scale study to evaluate the significance of SSTR2 immunohistochemistry in rectal NETs.However, we did not evaluate the value of SSTR2 expressions for SSTR-targeting PET/CT and PRRT in terms of clinical outcomes.Further studies with clinical value of PRRT or imaging modalities using SSTR expression is recommended to assess the overall effects of SSTR expressions in treatment of rectal NET patients.
In conclusion, approximately two-thirds of rectal NETs expressed SSTR2.SSTR2 expression were significantly associated with favorable behavior and good overall survival in patients with rectal NETs.Furthermore, SSTR2 expression can be used as prognostic factors.When metastatic disease occurs, SSTR2 expression can be used a possible target for somatostatin analogues.

Figure 3 .
Figure 3. Kaplan-Meier survival analyses of rectal neuroendocrine tumors.(a) The overall 5-year survival rate of patients with SSTR2 expression was significantly better than those without expression (98.5% vs. 92.6%,p = 0.006).(b) Patients with grade 1 rectal NET and SSTR2 expression had significant better overall 5-year survival rate than those without SSTR2 expression (98.4% vs. 93.4%,p = 0.011).(c) The overall 5-year survival rate of grade 2 rectal NET patients with SSTR2 expression was better than those without SSTR2 expression though statistically not significant (100.0%vs. 85.7%, p = 0.196).

Table 1 .
SSTR2a expression and correlation with clinicopathologic factors of rectal neuroendocrine tumors.

Table 2 .
Univariate and multivariate analyses.*Statistically significant at p < 0.05.a Endoscopically resected neuroendocrine tumors were regarded as not applicable cases.# All cases are censored so statistics are not calculated.SSTR somatostatin receptor, NA not applicable, AJCC American Joint Committee on Cancer, HR hazard ratio, CI confidence interval.